McDonald C D, Maher L J
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Nucleic Acids Res. 1995 Feb 11;23(3):500-6. doi: 10.1093/nar/23.3.500.
We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids. Homopyrimidine RNA oligonucleotides bind to duplex DNA at homopurine/homopyrimidine sequences under slightly acidic conditions. Recognition is sequence-specific, involving rU.dA.dT and rC+.dG.dC base triplets. Affinities were determined for folded polymeric RNAs (ca. 100-200 nt) containing 0, 1 or 3 copies of a 21 nt RNA sequence that binds duplex DNA by triple helix formation. When this recognition sequence was inserted into the larger folded RNAs, micromolar concentrations of the resulting RNA ligands bound a duplex DNA target at pH 5. However, these binding affinities were at least 20-fold lower than the affinity of an RNA oligonucleotide containing only the recognition sequence. Enzymatic probing of folded RNAs suggests that reduced affinity arises from unfavorable electrostatic, structural and topological considerations. The affinity of a polymeric RNA with three copies of the recognition sequence was greater than that of a polymeric RNA with a single copy of the sequence. This affinity difference ranged from 2.6- to 13-fold, depending on pH. Binding of duplex DNA by polymeric RNA might be improved by optimizing the RNA structure to efficiently present the recognition sequence.
我们对基于核酸对双链DNA的识别来创建人工基因阻遏物感兴趣。同嘧啶RNA寡核苷酸在略酸性条件下于同嘌呤/同嘧啶序列处与双链DNA结合。识别具有序列特异性，涉及rU.dA.dT和rC+.dG.dC碱基三联体。测定了含有0、1或3个通过三链螺旋形成与双链DNA结合的21 nt RNA序列拷贝的折叠多聚RNA（约100 - 200 nt）的亲和力。当将此识别序列插入较大的折叠RNA中时，所得RNA配体在pH 5时以微摩尔浓度结合双链DNA靶标。然而，这些结合亲和力比仅含有识别序列的RNA寡核苷酸的亲和力至少低20倍。对折叠RNA的酶促探测表明，亲和力降低是由于不利的静电、结构和拓扑因素。具有三个识别序列拷贝的多聚RNA的亲和力大于具有单个该序列拷贝的多聚RNA的亲和力。这种亲和力差异在2.6至13倍之间，取决于pH值。通过优化RNA结构以有效呈现识别序列，多聚RNA与双链DNA的结合可能会得到改善。
